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Characterizing standard genetic parts and establishing common principles for engineering legume and cereal roots.

ABSTRACT: Plant synthetic biology and cereal engineering depend on the controlled expression of transgenes of interest. Most engineering in plant species to date has relied heavily on the use of a few, well-established constitutive promoters to achieve high levels of expression; however, the levels of transgene expression can also be influenced by the use of codon optimization, intron-mediated enhancement and varying terminator sequences. Most of these alternative approaches for regulating transgene expression have only been tested in small-scale experiments, typically testing a single gene of interest. It is therefore difficult to interpret the relative importance of these approaches and to design engineering strategies that are likely to succeed in different plant species, particularly if engineering multigenic traits where the expression of each transgene needs to be precisely regulated. Here, we present data on the characterization of 46 promoters and 10 terminators in Medicago truncatula, Lotus japonicus, Nicotiana benthamiana and Hordeum vulgare, as well as the effects of codon optimization and intron-mediated enhancement on the expression of two transgenes in H. vulgare. We have identified a core set of promoters and terminators of relevance to researchers engineering novel traits in plant roots. In addition, we have shown that combining codon optimization and intron-mediated enhancement increases transgene expression and protein levels in barley. Based on our study, we recommend a core set of promoters and terminators for broad use and also propose a general set of principles and guidelines for those engineering cereal species.

SUBMITTER: Feike D

PROVIDER: S-EPMC6835126 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Characterizing standard genetic parts and establishing common principles for engineering legume and cereal roots.

Feike Doreen D Korolev Andrey V AV Soumpourou Eleni E Murakami Eiichi E Reid Dugald D Breakspear Andrew A Rogers Christian C Radutoiu Simona S Stougaard Jens J Harwood Wendy A WA Oldroyd Giles E D GED Miller J Benjamin JB

Plant biotechnology journal 20190523 12

Plant synthetic biology and cereal engineering depend on the controlled expression of transgenes of interest. Most engineering in plant species to date has relied heavily on the use of a few, well-established constitutive promoters to achieve high levels of expression; however, the levels of transgene expression can also be influenced by the use of codon optimization, intron-mediated enhancement and varying terminator sequences. Most of these alternative approaches for regulating transgene expre ...[more]

PMID: 31022324

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Project description:Cropping system diversification through annual intercropping provides a pathway for agricultural production with reduced inputs of fertilizer and pesticides. While several studies have shown that intercrop performance depends on the genotypes used, the available evidence has not been synthesized in an overarching analysis. Here, we review the effects of genotypes in cereal/legume intercropping systems, showing how genotype choice affects mixture performance. Furthermore, we discuss the mechanisms underlying the interactions between genotype and cropping system (i.e., sole cropping vs. intercropping). Data from 69 articles fulfilling inclusion criteria were analyzed, out of which 35 articles reported land equivalent ratio (LER), yielding 262 LER data points to be extracted. The mean and median LER were 1.26 and 1.24, respectively. The extracted genotype × cropping system interaction effects on yield were reported in 71% out of 69 publications. Out of this, genotype × cropping system interaction effects were significant in 75%, of the studies, whereas 25% reported non-significant interactions. The remaining studies did not report the effects of genotype × cropping system. Phenological and morphological traits, such as differences in days to maturity, plant height, or growth habit, explained variations in the performance of mixtures with different genotypes. However, the relevant genotype traits were not described sufficiently in most of the studies to allow for a detailed analysis. A tendency toward higher intercropping performance with short cereal genotypes was observed. The results show the importance of genotype selection for better in cereal/legume intercropping. This study highlights the hitherto unrevealed aspects of genotype evaluation for intercropping systems that need to be tackled. Future research on genotype effects in intercropping should consider phenology, root growth, and soil nutrient and water acquisition timing, as well as the effects of weeds and diseases, to improve our understanding of how genotype combination and breeding may help to optimize intercropping systems.

Project description:Intercropping legume with cereal is an extensively applied planting pattern in crop cultivation. However, forage potential and the degradability of harvested mixtures from intercropping system remain unclear. To investigate the feasibility of applying an intercropping system as a forage supply source to ruminants, two consecutive experiments (experiments 1 and 2) involving a field cultivation trial and a subsequent in vivo degradable experiment were conducted to determine the forage production performance and the ruminally degradable characteristics of a harvested mixture from an alfalfa/corn-rye intercropping system. In experiment 1, the intercropping system was established by alternating alfalfa and corn or rye with a row ratio of 5:2. Dry matter (DM) and nutrient yields were determined. In experiment 2, forages harvested from the different treatments were used as feedstuff to identify nutrient degradation kinetics and distribution of components between the rapidly degradable (a), potentially degradable (b) and the degradation rate constant (c) of 'b' fraction by in sacco method in Small-Tail Han wether Sheep. The intercropping system of alfalfa and corn-rye provided higher forage production performance with net increases of 9.52% and 34.81% in DM yield, 42.13% and 16.74% in crude protein (CP) yield, 25.94% and 69.99% in degradable DM yield, and 16.96% and 5.50% in degradable CP yield than rotation and alfalfa sole cropping systems, respectively. In addition, the harvest mixture from intercropping system also had greater 'a' fraction, 'b' fraction, 'c' values, and effective degradability (E value) of DM and CP than corn or rye hay harvested from rotation system. After 48-h exposure to rumen microbes, intercropping harvest materials were degraded to a higher extent than separately degraded crop stems from the sole system as indicated by visual microscopic examination with more tissues disappeared. Thus, the intercropping of alfalfa and corn-rye exhibited a greater forage production potential, and could be applied as forage supply source for ruminants. The improved effective degradability of harvest mixture material could be attributed to greater degradable components involving the rapidly degradable fractions (a), potentially degradable (b) fractions, and degradable rate constant

Project description:BACKGROUND: Modularity is a crucial issue in the engineering world, as it enables engineers to achieve predictable outcomes when different components are interconnected. Synthetic Biology aims to apply key concepts of engineering to design and construct new biological systems that exhibit a predictable behaviour. Even if physical and measurement standards have been recently proposed to facilitate the assembly and characterization of biological components, real modularity is still a major research issue. The success of the bottom-up approach strictly depends on the clear definition of the limits in which biological functions can be predictable. RESULTS: The modularity of transcription-based biological components has been investigated in several conditions. First, the activity of a set of promoters was quantified in Escherichia coli via different measurement systems (i.e., different plasmids, reporter genes, ribosome binding sites) relative to an in vivo reference promoter. Second, promoter activity variation was measured when two independent gene expression cassettes were assembled in the same system. Third, the interchangeability of input modules (a set of constitutive promoters and two regulated promoters) connected to a fixed output device (a logic inverter) expressing GFP was evaluated. The three input modules provide tunable transcriptional signals that drive the output device. If modularity persists, identical transcriptional signals trigger identical GFP outputs. To verify this, all the input devices were individually characterized and then the input-output characteristic of the logic inverter was derived in the different configurations. CONCLUSIONS: Promoters activities (referred to a standard promoter) can vary when they are measured via different reporter devices (up to 22%), when they are used within a two-expression-cassette system (up to 35%) and when they drive another device in a functionally interconnected circuit (up to 44%). This paper provides a significant contribution to the study of modularity limitations in building biological systems by providing useful data on context-dependent variability of biological components.

Dataset Information

Characterizing standard genetic parts and establishing common principles for engineering legume and cereal roots.

Publications

Characterizing standard genetic parts and establishing common principles for engineering legume and cereal roots.

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